The multicatalytic proteinase or the proteasome is a highly conserved cellular structure that is responsible for the ATP-dependent proteolysis of most cellular proteins (Coux. O. Tanaka, K. and Goldberg, A. 1996 Ann. Rev. Biochem. 65, 801-847). The 20S proteasome contains the catalytic core of the complex and has been crystallized from the archaebacteria Thermoplasma acidophilum (Lowe, J., Stock. D., Jap, B., Zwicki, P., Bauminster, W. and Huber, R. 1995 Science 268, 533-539) and from the yeast Saccharomyces cerevisiae (Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik, HD and Huber, R. 1997 Nature 386, 463-471). Unlike the archaebacterial proteasome that primarily exhibits chymotrypsin-like proteolytic activity (Dahlmann, B., Kopp, F., Kuehn, L., Niedel, B., Pfeifer, G. 1989 FEBSLett. 251, 125-131; Seemuller, E., Lupas, A., Zuw, F., Zwickl, P and Baumeister, W. FEBS Lett. 359, 173, (1995) the eukaryotic proteasome contains at least five identifiable proteolytic activities. Three of these activities are similar in specificity to chymotrypsin, trypsin and peptidylglutamyl peptidase. The two other activities described exhibit a preference for cleavage of peptide bonds on the carboxyl side of branched chain amino acids (BrAAP) and toward peptide bonds between short chain neutral amino acids (SnAAP) (Orlowski, M. 1990 Biochemistry 29, 10289-10297).
Although the 20S proteasome contains the proteolytic core, it cannot degrade proteins in vivo unless it is complexed with a 19S cap, at either end of its structure, which itself contains multiple ATPase activities. This larger structure is known as the 26S proteasome and will rapidly degrade proteins that have been targeted for degradation by the addition of multiple molecules of the 8.5-kDa polypeptide, ubiquitin (reviewed in Coux, O., Tanaka, K. and Goldberg, A. 1996 Ann.Rev. Biochem. 65, 801-847).
A large number of substrate-derived functionalities have been used as potential serine-and thiol protease inhibitors. Several of these motifs have been described as inhibitors to the proteasome. These include the peptide aldehydes (Vinitsky. A.. Michaud. C., Powers, J. and Orlowski. M. 1992 Biochemistry 31, 9421-9428; Tsubuki. S., Hiroshi, K. Saito, Y., Miyashita, N., Inomata, M., and Kawashima, S. 1993 Biochiem.Biophys.Res.Commun. 196,1195-1201; Rock, K, I., Gramm, C., Rothstein, L., Clark, K., Stein, R., Dick, L., Hwang, D. and Goldberg, A. L. (1994) Cell 78, 761-771) N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) and N-acetyl-L-leucinyl-1-leucinyl-methional (LLM) with the most potent inhibitor of this type being N-carbobenzoxyl-1-L-leucinyl-L-leucinyl-L-norvalinal (MG115). Other reports have described a series of dipeptide inhibitors that have IC50 values in the 10 to 100 nM range (Iqbal, M., Chatterjee S., Kauer, J. C., Das, M., Messina, P., Freed, B., Biazzo, W and Siman, R. 1995 I-Med.Chem.38, 2276-2277). A series of xcex1-ketocarbonyl and boronic ester derived dipeptides (Iqbai, M., Chatterjee, S., Kauer, J. C., Mallamo, J. P., Messina, P. A., Reiboldt, A. and Siman, R. 1996 Bioorg. Med-Chem. Lett 6, 287-290) and epoxyketones (Spattenstein, A., Leban, J J., Huang, J. J., Reinhardt, K. R., Viveros, O. H., Sigafoos, J. and Crouch, R. 1996 Tet. Lett. 37, 1434-1346) have also been described that are potent inhibitors of the proteasome.
A different compound that exhibits specificity in inhibiting proteasome activity is Lactacystin (Fenteany, G., Standaert, R. F., Lane, W. S., Choi, S., Corey, E. J. and Schreiber, S. L. 1995 Science 268, 726-731) which is a Streptomyces metabolite. This molecule was originally discovered for its ability to induce neurite outgrowth in a neuroblastoma cell line (Omura, S., Matsuzaki, K., Fujimoto, T., Kosuge, K., Furuya, T., Fujita, S. and Nakagawa, A. 1991 J.Antibiot. 44, 117-118) and later was shown to inhibit the proliferation of several cell types (Fenteany. G., Standaert. R. F., Reichard, G. A., Corey, E. J. and Schreiber, S. L.1994 Proc.Nat""l. Acad.Sci. USA 91. 3358-3362).
It is now well established that the proteasome is a major extralysosomal proteolytic system involved in the proteolytic pathways essential for diverse cellular functions such as cell division, antigen processing and the degradation of short-lived regulatory proteins such as oncogene productsm, cyclins and transcription factors (Ciechanover, A. (1994) Cell 79, 13-21;Palombell, V. J., Rando, O. J., Goldberg, A. L. and Maniatis, T. 1994 Cell 78, 773-785). For example, the active form of NF-xcexaB is a heterodimer consisting of a p65 and a p50 subunit. The latter is present in the cytosol as an inactive precursor (p105). The proteolytic processing of p105 to generate p50 occurs via the ubiquitin-proteasome pathway. Additionally, processed p50 and p65 are maintained in the cytosol as an inactive complex bound to the inhibitory protein IxcexaB. Inflammatory stimuli such as LPS activate NF-xcexaB by initiating the signalling pathway which leads to the degradation of IxcexaB. These signals also stimulate the processing of p105 into p50. Thus two proteolytic events, both governed by the ubiquitin-proteasome pathway, are required for signal induced activation of NF-xcexaB.
The observation that ubiquitin-mediated proteasomal proteolysis plays a critical role in the activation of NF-xcexaB could be exploited clinically by the use of inhibitors directed toward the proteasome. Abnormal activation of NF-xcexaB followed by the stimulation of cytokine synthesis has been observed in a variety of inflammatory and infectious diseases. Activation of NF-xcexaB is also essential for angiogenesis and for expression of adhesion molecules (CAMs and selects), thus proteasome inhibitors may also have utility in the treatment of diseases associated with the vascular system.
It is well documented that the ubiquitin-proteasome pathway is critical for the regulated destruction of cyclins that govern the exit from mitosis and allow cells to progress into the next phase of the cell cycle (Glotzer, M., Murray, A. W. and Kirschner, M. W. (1991) Nature 349, 132-138). Thus, inhibiting the degradation of cyclins by using proteasome inhibitors causes growth arrest. Therefore another potential utility of proteasome inhibitors is their use in the treatment of diseases that result from abberrant cell division.
Several classes of peptidic inhibitors of 20S proteasome have been reported in the recent literature. The xcex1-ketoamide group has been used in protease inhibitors for numerous indications. Specifically, a series of xcex1-ketocarbonyl and boronic ester derived dipeptides (Iqbal, M., Chatterjee, S., Kauer, J. C., Mallamo, J. P., Messina, P. A., Reiboldt, A. and Siman, R. 1996 Bioorg. Med.Chem. Lett 6, 287-290) have been reported as potent inhibitors of 20S proteasomal function. Derivatives of 3-indolepyruvic acid have been claimed as pharmaceutically active compounds for the treatment of disturbances of the central nervous system (De Luca, et al WO 88/09789) through a mechanism that modulates kynurenic acid levels in the brain.
Even though various compositions have been discovered that inhibit cell proliferation to some degree, there remains a need for more potent compounds that inhibit cell proliferation via the 20S proteasome.
It is an object of this invention to provide a method for inhibiting cell proliferation in mammals that uses a therapeutically effective amount of a composition heretofore unknown for its cell proliferative inhibition properties.
It is also an object of this invention to provide a method for the treatment of diseases that may be affected by the inhibition of proteosomal function.
Further, it is an object of this invention to provide a method for the treatment of proliferative diseases that operates by inhibiting proteasomal function.
It is another object of this invention to use a therapeutically effective amount of the compositions described herein to inhibit cell proliferative disorders in humans.
Yet another object of this invention is the use of a therapeutically effective amount of the compositions described herein to inhibit proteasomal function.
In one embodiment, this invention is a composition of matter having the formula: 
wherein X2 is Ar or Arxe2x80x94X3 wherein X3 is xe2x80x94Cxe2x95x90O, or xe2x80x94CH2COxe2x80x94, and wherein Ar is phenyl, substituted phenyl, indole, substituted indoles, and any other heteroaryls; R1, and R2 are each individually selected from the side chains of the known natural xcex1-amino acids and unnatural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl, substituted aryl, 1-10 carbon linear, branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle substituted heterocycle, heteroaryl and substituted heteroaryl; X1 is selected from hydroxide, monoalkylamino, dialkylamino, alkoxide, arylkoxide and 
wherein X4 is hydroxide, arylamino, monoalkylamino, dialkylamino, alkoxide, or arylalkoxide; and
R3 is selected from the known natural xcex1-amino acids, unnatural amino acids, hydrogen, 1-10 carbon linear and branched alkyl, 1-10 carbon linear and branched substituted alkyl, aryl, substituted aryl, 1-10 carbon linear and branched substituted aryl, alkoxyaryl, 3-8 carbon cycloalkyl, heterocycle, substituted heterocycle, heteroaryl and substituted heteroaryl.
In another embodiment, this invention is a method for inhibiting proteasomal protease factor in mammals comprising administering a therapeutically effective amount of the composition disclosed above to the mammal.
In still another embodiment, this invention is a pharmaceutical composition of matter comprising the composition of claim 1 and one or more pharmaceutical excipients.